The primary objectives of this project are to further our understanding of the mechanism of excitability in endocrine cells and to determine the mechanism by which stimulators or inhibitors of hormone secretion alter the electrical activity of the cells they act upon. To accomplish these goals, macroscopic and single channel current measurements, made with the patch clamp technique, will be used to identify and characterize the membrane conductances responsible for excitability in these cells. In particular, we will focus on pancreatic islet cells, by studying both glucagon-secreting A-cells and insulin-secreting B-cells. The preparations we will utilize include a clonal line of B-cells (HIT cells), and normal adult islet cells isolated from the rat pancreas. A- or B-cells will be identified from the hormone they release, by making use of the reverse hemolytic plaque assay. Channel identification will be determined primarily by studying ion selectivity, pharmacology, single channel conductance and kinetics. Following these preliminary experiments, we will study the mechanisms by which agents that affect secretion alter the membrane potential of the cells they act upon, for example glucose on B-cells. In the case of A-cells, we will first describe any changes in A-cell electrical activity that accompany a secretory stimulus, such as a reduction in glucose concentration or the presentation of arginine. In addition to the primary secretagogues we will also examine the effects of neurotransmitters, such as acetylcholine and norepinephrine, on the ionic channels found in A- or in B-cells. We will determine if any of these agents activate ionic channels in a manner similar to acetylcholine at the neuromuscular junction, or if they alter, either directly or indirectly, the properties of existing channels. This information will hopefully lead to a better understanding of the mechanism of stimulus-secretion coupling in endocrine cells, and its modulation by activity of the autonomic nervous system. A final objective of this proposal is to perform certain biophysical studies of channel function, utilizing channels found in our islet cell preparations. One such study would examine the role of permeant ions and calcium on potassium channel gating at the single channel level.